Intracellular GM-CSF signaling in circulating monocytes, monocyte-derived macrophages, and human lung macrophages

<bold>Introduction:</bold> GM-CSF binds to its receptor, activating JAK2 and phosphorylation of the transcription factor STAT5. We compared the GM-CSF signaling in circulating monocytes (MOs), monocyte-derived macrophages (MDMs) and human lung macrophages (LMs). <bold>Methods:</bold> LMs were sorted after digestion of human lung tissue. MDMs were obtained after 6 days of culture of MOs in the presence of GM-CSF (50 ng/ml) and a 24hours-period starving. Cells were stimulated with GM-CSF (10 or 50 ng/ml) before being fixed, permeabilized, labeled (CD45, HLA-DR, CD43, CD206, CD14, CD16, pSTAT5) and analyzed on a CytoFlex SRT flow cytometer (Beckman Coulter©). pSTAT5 was also revealed by Western blot and electrochemiluminescence (MSD technology©) from MOs, MDMs and LMs lysates. Presence of GM-CSF receptor was studied by confocal microscopy on MOs and LMs. <bold>Results:</bold> GM-CSFR chains were expressed by MOs and LMs. Stimulation with GM-CSF induced a significant increase in the proportion of pSTAT5+ cells in MOs but did not in MDMs or LMs. Among the cellular fraction from lung tissue treated with GM-CSF, induction of pSTAT5 occurred in monocytes but not in alveolar or interstitial macrophage. In vitro, pSTAT5 induction by GM-CSF was observed in MOs, but decreased during the process of differentiation into MDMs and was no longer identifiable after the 7 days of culture. In Western blot and MSD experimentations, GM-CSF (10 ng/ml, 10 min) induced pSTAT5 at a twofold level in MOs compared to MDMs and LMs. <bold>Conclusion:</bold> Intracellular GM-CSF signaling is down-regulated in fully differentiated lung macrophages, eventually explaining the rarity of pulmonary side effects reported in trials evaluating anti-GM-CSF therapies